Control strategies for integrated continuous bioprocessing

Continuous Bioprocessing is perceived to deliver constant product quality and to achieve higher time space yields. This is not new. Other market segments run well established continuous processes successfully since decades. What do we need to do? Just establishing continuous single unit operations (e.g. SMB chromatography) will not suffice. We need to have an integrated look at the entire process chain and stringently use the following control strategies : Capture process variability of the preceding unit operation using advanced PAT tools. Just measuring CPPs will not suffice: We need RMAs and intermediate CQAs to be transparent across the process chain. Include above variability in the control strategy of the individual unit operations, carry out sound process characterization using data science tools. Have clear specifications for intermediate acceptance criteria (IACs), such as distributions of expected outputs of intermediate CQAs Apply advanced process control strategies, such as multiple input multiple output controllers to robustly link process chain elements. Be aware that additional variability may occur along the life cycle. Hence, establish holistic manufacturing control strategies fulfilling Established Conditions (ECs) along ICH Q12 rationales. Please click Additional Files below to see the full abstract

Data analytics and soft sensors for single use bioprocessing

In the last decades, innovative research and engineering brought birth to a plethora of robust and mature process analytical devices. Nowadays, the possibilities to extract chemical-, physical- and biological data from single use processes are manifold and a great quantity of process data is collected on a routine basis. However, novel challenges in the field of data processing and information mining emerged: How can the maximum information content be extracted from the combination of process analyzers? How can big process data be handled and exploited efficiently? And ultimately: How can this information be translated in a business benefit for the manufacturers? Here, we demonstrate how these challenges can be addressed within the bioprocess lifecycle using innovative mechanistic methods. We present i) novel non-invasive soft sensors for real-time monitoring of single use processes, ii) information mining and process analysis based on the combination of mechanistic models and statistical tools and iii) efficient and scalable process control strategies. Financial support was provided by the Austrian research funding association (FFG) under the scope of the COMET program within the research network “Process Analytical Chemistry (PAC)” (contract # 825340). This programme is promoted by BMVIT, BMWFJ and the federal state of Upper Austria

A Method for Finding a Design Space as Linear Combinations of Parameter Ranges for Biopharmaceutical Control Strategies

According to ICH Q8 guidelines, the biopharmaceutical manufacturer submits a design space (DS) definition as part of the regulatory approval application, in which case process parameter (PP) deviations within this space are not considered a change and do not trigger a regulatory post approval procedure. A DS can be described by non-linear PP ranges, i.e., the range of one PP conditioned on specific values of another. However, independent PP ranges (linear combinations) are often preferred in biopharmaceutical manufacturing due to their operation simplicity. While some statistical software supports the calculation of a DS comprised of linear combinations, such methods are generally based on discretizing the parameter space - an approach that scales poorly as the number of PPs increases. Here, we introduce a novel method for finding linear PP combinations using a numeric optimizer to calculate the largest design space within the parameter space that results in critical quality attribute (CQA) boundaries within acceptance criteria, predicted by a regression model. A precomputed approximation of tolerance intervals is used in inequality constraints to facilitate fast evaluations of this boundary using a single matrix multiplication. Correctness of the method was validated against different ground truths with known design spaces. Compared to stateof-the-art, grid-based approaches, the optimizer-based procedure is more accurate, generally yields a larger DS and enables the calculation in higher dimensions. Furthermore, a proposed weighting scheme can be used to favor certain PPs over others and therefore enabling a more dynamic approach to DS definition and exploration. The increased PP ranges of the larger DS provide greater operational flexibility for biopharmaceutical manufacturers.Comment: 15 pages, 7 figures, 3 tables, research articl

A dynamic method based on the specific substrate uptake rate to set up a feeding strategy for Pichia pastoris

<p>Abstract</p> <p>Background</p> <p><it>Pichia pastoris </it>is one of the most important host organisms for the recombinant production of proteins in industrial biotechnology. To date, strain specific parameters, which are needed to set up feeding profiles for fed batch cultivations, are determined by time-consuming continuous cultures or consecutive fed batch cultivations, operated at different parameter sets.</p> <p>Results</p> <p>Here, we developed a novel approach based on fast and easy to do batch cultivations with methanol pulses enabling a more rapid determination of the strain specific parameters specific substrate uptake rate q_s, specific productivity q_pand the adaption time (Δtime_adapt) of the culture to methanol. Based on q_s, an innovative feeding strategy to increase the productivity of a recombinant <it>Pichia pastoris </it>strain was developed. Higher specific substrate uptake rates resulted in increased specific productivity, which also showed a time dependent trajectory. A dynamic feeding strategy, where the setpoints for q_swere increased stepwise until a q_s_maxof 2.0 mmol·g^-1·h^-1resulted in the highest specific productivity of 11 U·g^-1·h^-1.</p> <p>Conclusions</p> <p>Our strategy describes a novel and fast approach to determine strain specific parameters of a recombinant <it>Pichia pastoris </it>strain to set up feeding profiles solely based on the specific substrate uptake rate. This approach is generic and will allow application to other products and other hosts.</p

Radial line-scans as representative sampling strategy in dried-droplet laser ablation of liquid samples deposited on pre-cut filter paper disks

Nebulising liquid samples and using the aerosol thus obtained for further analysis is the standard method in many current analytical techniques, also with inductively coupled plasma (ICP)-based devices. With such a set-up, quantification via external calibration is usually straightforward for samples with aqueous or close-to-aqueous matrix composition. However, there is a variety of more complex samples. Such samples can be found in medical, biological, technological and industrial contexts and can range from body fluids, like blood or urine, to fuel additives or fermentation broths. Specialized nebulizer systems or careful digestion and dilution are required to tackle such demanding sample matrices. One alternative approach is to convert the liquid into a dried solid and to use laser ablation for sample introduction. Up to now, this approach required the application of internal standards or matrix-adjusted calibration due to matrix effects. In this contribution, we show a way to circumvent these matrix effects while using simple external calibration for quantification. The principle of representative sampling that we propose uses radial line-scans across the dried residue. This compensates for centro-symmetric inhomogeneities typically observed in dried spots. The effectiveness of the proposed sampling strategy is exemplified via the determination of phosphorus in biochemical fermentation media. However, the universal viability of the presented measurement protocol is postulated. Detection limits using laser ablation-ICP-optical emission spectrometry were in the order of 40 mu g mL(-1) with a reproducibility of 10 % relative standard deviation (n = 4, concentration = 10 times the quantification limit). The reported sensitivity is fit-for-purpose in the biochemical context described here, but could be improved using ICP-mass spectrometry, if future analytical tasks would require it. Trueness of the proposed method was investigated by cross-validation with conventional liquid measurements, and by analyzing IAEA-153 reference material (Trace Elements in Milk Powder); a good agreement with the certified value for phosphorus was obtaine

Microbials for the production of monoclonal antibodies and antibody fragments

Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli

Identification of lipophilic bioproduct portfolio from bioreactor samples of extreme halophilic archaea with HPLC-MS/MS

Extreme halophilic archaea are a yet unexploited source of natural carotenoids. At elevated salinities, however, material corrosivity issues occur and the performance of analytical methods is strongly affected. The goal of this study was to develop a method for identification and downstream processing of potentially valuable bioproducts produced by archaea. To circumvent extreme salinities during analysis, a direct sample preparation method was established to selectively extract both the polar and the nonpolar lipid contents of extreme halophiles with hexane, acetone and the mixture of MeOH/MTBE/water, respectively. Halogenated solvents, as used in conventional extraction methods, were omitted because of environmental considerations and potential process scale-up. The HPLC-MS/MS method using atmospheric pressure chemical ionization was developed and tuned with three commercially available C-40 carotenoid standards, covering the wide polarity range of natural carotenoids, containing different number of OH-groups. The chromatographic separation was achieved on a C-30 RP-HPLC column with a MeOH/MTBE/water gradient. Polar lipids, the geometric isomers of the C-50 carotenoid bacterioruberin, and vitamin MK-8 were the most valuable products found in bioreactor samples. In contrast to literature on shake flask cultivations, no anhydrous analogues of bacterioruberin, as by-products of the carotenoid biosynthesis, were detected in bioreactor samples. This study demonstrates the importance of sample preparation and the applicability of HPLC-MS/MS methods on real samples from extreme halophilic strains. Furthermore, from a biotechnological point-of-view, this study would like to reveal the relevance of using controlled and defined bioreactor cultivations instead of shake flask cultures in the early stage of potential bioproduct profiling

Adhesion molecules in different treatments of acute myocardial infarction

BACKGROUND: Tissue damage after ischemia and reperfusion involves leukocyte endothelial interactions mediated by cell adhesion molecules. This study was designed to determine the time course of soluble adhesion molecules in patients with acute myocardial infarction after attempted reperfusion by thrombolysis with tissue plasminogen activator (tPA) or streptokinase (SK), or percutaneous transluminal coronary angioplasty (PTCA). METHODS: In 3 × 10 randomly selected patients with acute myocardial infarction undergoing thrombolysis with tPA or SK, or treated with PTCA, plasma concentrations of soluble L-selectin, P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) were measured by enzyme-linked immunosorbent assay, 30 min and 1, 2, 4, 8, 12 and 24 hours after intervention. RESULTS: After thrombolysis with tPA, soluble L-selectin concentrations were persistently depressed and soluble PECAM-1 concentrations were elevated, compared with controls, SK and PTCA. While soluble VCAM-1 concentrations did not differ within the first hours after interventions between the three groups, soluble VCAM-1 rose by 24 hours after tPA thrombolysis but did not increase after SK and PTCA treatment. Soluble ICAM-1 concentrations were consistently elevated after PTCA compared with controls and thrombolysed patients. Soluble E-selectin was depressed after tPA thrombolysis and PTCA in comparison with controls, while the SK group showed an increase throughout the observation period. Soluble P-selectin was increased after PTCA and SK lysis up to 8 hours after treatment compared with controls, but no significant differences could be found between treatment groups. CONCLUSION: Adhesion molecules mediating leukocyte endothelial interactions are altered subsequent to postischemic reperfusion and by treatment with thrombolytic agents and angioplasty. The clinical relevance of these biological changes remains to be determined

Selection for atrial fibrillation ablation: Importance of diastolic function grading

AbstractBackgroundPulmonary vein isolation (PVI) has become an accepted therapy for patients with atrial fibrillation (AF) and the indications have widened to include non-paroxysmal AF-patients. Maintenance of sinus rhythm after PVI can be adversely affected by clinical or echocardiographic parameters, which should be clearly identified.Methods and resultsAfter baseline clinical and echocardiographic evaluations, PVI was performed in patients with paroxysmal or non-paroxysmal AF. The follow-up strategy after PVI included: (1) clinical follow up, 12-lead electrocardiography (ECG) and 24-h ECG every 3 months, (2) trans-telephonic ECGs twice daily and when symptomatic (over 4 weeks) every 3 months, or (3) continuous monitoring via implanted devices. A recurrence was an atrial arrhythmia lasting >30s. All 340 PVI procedures of 229 patients were analyzed. On average, 1.5 PVI procedures per patient (range, 1–6 PVI) were performed. The mean age was 58±11 years (73% male) with 109 paroxysmal and 120 non-paroxysmal AF cases. Clinical follow-up with 12-lead ECGs, 24-h ECGs, trans-telephonic ECGs, and implanted devices was complete in 100%, 63%, 51%, and 16% of cases, respectively. The overall one-year recurrence rate of 59% (range, 24–82%) was dependent on grades of diastolic function (normal – dysfunction grade III) in a multivariable analysis model. Patients with normal diastolic function had the lowest recurrence rates of 24% and 49% after 1 and 3 years of follow-up, respectively (p<0.0001).ConclusionDiastolic function could serve as a simple summary predictor for AF recurrence, and would facilitate clinical decision-making in AF treatment